Chitosan‐based nanoscale systems for doxorubicin delivery: Exploring biomedical application in cancer therapy

Abstract Green chemistry has been a growing multidisciplinary field in recent years showing great promise in biomedical applications, especially for cancer therapy. Chitosan (CS) is an abundant biopolymer derived from chitin and is present in insects and fungi. This polysaccharide has favorable characteristics, including biocompatibility, biodegradability, and ease of modification by enzymes and chemicals. CS‐based nanoparticles (CS‐NPs) have shown potential in the treatment of cancer and other diseases, affording targeted delivery and overcoming drug resistance. The current review emphasizes on the application of CS‐NPs for the delivery of a chemotherapeutic agent, doxorubicin (DOX), in cancer therapy as they promote internalization of DOX in cancer cells and prevent the activity of P‐glycoprotein (P‐gp) to reverse drug resistance. These nanoarchitectures can provide co‐delivery of DOX with antitumor agents such as curcumin and cisplatin to induce synergistic cancer therapy. Furthermore, co‐loading of DOX with siRNA, shRNA, and miRNA can suppress tumor progression and provide chemosensitivity. Various nanostructures, including lipid‐, carbon‐, polymeric‐ and metal‐based nanoparticles, are modifiable with CS for DOX delivery, while functionalization of CS‐NPs with ligands such as hyaluronic acid promotes selectivity toward tumor cells and prevents DOX resistance. The CS‐NPs demonstrate high encapsulation efficiency and due to protonation of amine groups of CS, pH‐sensitive release of DOX can occur. Furthermore, redox‐ and light‐responsive CS‐NPs have been prepared for DOX delivery in cancer treatment. Leveraging these characteristics and in view of the biocompatibility of CS‐NPs, we expect to soon see significant progress towards clinical translation.

the mechanisms that can lead to cancer drug resistance. [11][12][13][14] Given the importance of drug resistance in chemotherapy failure, scientists have followed some strategies for overcoming this condition by applying nanostructures that improve drug delivery potential, enhance intracellular accumulation, and provide targeted delivery and codelivery with other antitumor agents or nucleic acid therapeutics. 15,16 The aim of present review is to discuss the role of chitosan (CS) for the delivery of DOX as one of the most well-known chemotherapeutic agents in cancer therapy and introducing CS chemistry, structure, and potential applications in medicine. The function of DOX in cancer suppression, factors responsible for its resistance and role of nanoparticles in reversing DOX resistance are discussed with emphasis on CS-based nanostructures for its delivery; pH-and redox-sensitive assorted CS nanoparticles are highlighted including their use for co-delivery of DOX with antitumor agents and nucleic acid therapeutics. Finally, the modification of various nanoparticles and appropriate solutions for their clinical applications are described to shed a light on the deployment of these nanostructures for cancer chemotherapy.

| CHITOSAN: CHEMISTRY AND BIOMEDICAL APPLICATION
The green technology is one of the newest and most recent approaches for the development of nanopharmaceuticals in treatment of diseases.
The green chemistry approach utilizes compounds and agents derived from nature for synthesis and modification of nanocarriers to improve their characteristics and make them better options for disease treatment. In this strategy, the hazardous material application is avoided and in turn, safe, biorenewable and biocompatible agents isolated from nature are utilized to develop nanoparticles. Since green-based nanocarriers demonstrate good safety profile and biocompatibility, the way for their clinical application is paved. The delivery of drugs and nucleic acid therapeutics is essential in cancer therapy due to their low accumulation at tumor site and emergence of drug resistance; hence, green synthesis or green modification of nanoparticles can be beneficial in this case for improving efficacy in cancer therapy. [17][18][19] In the present review, our aim is to highlight greener modifications of nanoparticles with CS as a natural compound to show its potential for cancer chemotherapy and possible clinical applications in the near future.
After cellulose, chitin is the most abundant natural polymer 20 and CS is derived from chitin (Figure 1), 22 an essential component comprising shells of insects, crustaceans, and cell walls of fungi. [23][24][25] The annual production of chitin is estimated to be 10-100 Gt and the commercialized chitin/CS can result from seafood waste in which αand β-chitins are derived from shells of crab and shrimp, while CS is prepared by deacetylation of chitin. 23,24,[26][27][28] The amount of deacetylation seems to be more than 60% in commercialized CS where Japan is considered as the major producer of CS. Based on the estimates, the value of CS market has been $6.8 B$ in 2019. 20 The unique chemical structure of CS has made it a suitable option for biomedical and engineering applications. The most important feature of CS is its great solubility in aqueous solution due to the presence of amino groups at C2 position. In aqueous acidic solvents, CS undergoes protonation to generate NH 3 + that is beneficial in the design of nanoarchitectures and their synthesis via bottom-up approach. Importantly, amino and acetylamino groups in CS are main sources of nitrogen for generating fertilizers and N-doped carbon materials for deployment as catalyst. 20,29 The application of CS in industry has demonstrated potential in reducing environmental pollution as a biodegradable and renewable abundant material that should not be discarded in to scarce landfills.
The aim of green chemistry is to limit industrial production of hazardous compounds and prevent destructive impacts, both short-term and long-term, on ecosystem. 30 [41][42][43] The biocompatibility and biodegradability are other beneficial characteristics of CS. [44][45][46] The biomedical application of CS nanostructures 47 has garnered much attention in recent years, especially in cancer therapy, which has been investigated in detail; CS nanoparticles (CS NPs) can mediate drug and nucleic acid therapeutic delivery, 48 chemotherapy, 49 phototherapy, 50 and imaging in cancer treatment. 51 The redox-sensitive micelles with carboxymethyl CS decoration can be utilized for NIR imaging of liver cancer cells and simultaneously, photo-and chemo-therapy. 49,52 Another study evaluated the potential of gold (Au)-embedded CS nanostructures for delivery of drugs in a pH-sensitive manner and providing fluorescence imaging. 53

| DOXORUBICIN: MECHANISM OF ACTION AND RESISTANCE
The DOX is an anthracycline antibiotic derived from Streptomyces peucetius caesius with high antitumor activity 77-79 as it displays efficacy even at low doses in suppressing different neoplasms. 80 The animal experiments evaluating anticancer activity of DOX have affirmed its potential in minimizing tumor progression and improving survival of animal models. 81  The CS is a pH-sensitive agent due to the presence of amine groups ( NH 2 ) that undergoes protonation in acidic pH 108,109 ; higher pH significantly decreases the solubility of CS. 109 On the other hand, polyvinylpyrrolidone (PVP) is often utilized for the synthesis of nanoparticles, but it significantly decreases the initial burst release. 110 For overcoming such issues, the combination of PVP and CS has been suggested to improve the solubility of CS at high pH levels and mechanical characteristic of PVP, simultaneously. 109 Figure 3). 120 The UV-triggered injectable CS hydrogels are extensively applied in biomedicine. 121 129 The purpose of using CS in the modification of nanoparticles is its capacity in functionalizing or loading various antitumor drugs (DOX), targeting ligands (aptamer), coating polymers and imaging probes. 130  The entrapment efficiency was 90% and after loading liposomal DOX in the hydrogel, its entrapment efficiency did not change. 149 Notably, carbon nanotube (CNT)-CS can be loaded in thermosensitive hydrogels for controlling DOX release. Their exposure to irradiation provides a photothermal effect of CNTs that is beneficial in destroying hydrogel structure and mediating DOX delivery. 150 Therefore, a thermosensitive hydrogel can be synthesized first for conversion to solid gel at body temperature and in the next step, CS-carbon nanotubes are loaded into the hydrogel for regulating DOX release upon irradiation. 150 The succeeding section focuses on multisensitive CS-based nanocarriers for DOX delivery.

| Multiresponsive
There have been many efforts in developing multifunctional CS-based  Table 1). [159][160][161] Besides internal-responsive CS-NPs for DOX delivery, there is another category termed hyperthermia-based external-stimuli drug delivery systems. These systems are responsive to an external stimulus, like magnetic field and light capable of increasing the temperature of the tumor microenvironment followed by killing the cancerous cells. 162 Moreover, after being triggered by the mentioned stimuli agents, the anticancer drug release rate undergoes a significant increase thus improving the efficiency of the delivery system. 163 This strategy has been exemplified by a multifunctional chemo-

| Doxorubicin and gene delivery
Gene therapy is a new emerging field for disease management and it can be considered as an option for treatment of diseases that are incurable with conventional medicines. 174 Figure 7).  Although CS has a positive charge and can form stable complexes with genes possessing a negative charge, it has a hydrophilic segment that reduces its affinity toward genes and shows poor solubility. 192 To overcome these drawbacks, a dendronized CS derivative (poly  194,195 The CS has been considered as a promising agent for modification of NPs for the delivery of small interfering RNA (siRNA) due to its positive charge, capacity in generating stable complex, high biocompatibility, and effective suppression of tumor cells. 177 The interest for the delivery of siRNA is due to circumventing a number of challenges such as poor internalization in tumor cells, prolonging blood circulation and preventing siRNA degradation by RNase enzymes. 13,[196][197][198] The CS-based nanostructures can provide a platform for the co-

| Doxorubicin and antitumor drug delivery
In addition to gene therapy as a promising strategy in promoting cytotoxicity of DOX against tumor cells, there have been attempts to recognize drugs that can exert synergistic impact with DOX in tumor suppression. The most well-known mechanism is that a certain antitumor agent induces DNA damage and apoptosis in cancer cells and then, the pathway is paved for DOX to inhibit tumor cell growth and invasion. 217,218 This section focuses on DOX and drug co-delivery by CS NPs in cancer therapy.
Lung cancer is a leading cause of death worldwide and based on new estimates, it is the most common cancer in both males and females. 219

| Lipid nanoparticle modification
Liposomes are synthetic lipid NPs that have been first discovered in the 1960s and comprise a lipid bilayer with an aqueous core. 230 The The DOX-loaded micelles can be prepared using alginate and CS in a water-in-oil emulsion method with a spherical particle size of 80 nm. This is an interesting method for loading DOX in nanocarriers and uses an aqueous phase dispersed in a cyclohexane/dodecylamine organic phase. These nanocarriers showed high cellular uptake by breast cancer cells and can suppress proliferation of 4 T1 cells. 243 The CS-modified micelles can also provide co-delivery of DOX and curcumin in liver cancer therapy. The CS-cystamine-poly(ε-caprolactone) copolymer micelles have been prepared for curcumin and DOX codelivery and then, modification with GA has been performed in enhancing their cellular uptake. They showed drug loading efficiency of 19.8% and 8.9% for DOX and curcumin, respectively. They had a spherical shape with a particle size of 110 nm. The GA modification       Figure 7 demonstrates modification of carbon-based nanomaterials with CS and then, conjugation of folic acid as ligand on nanocarriers to mediate their internalization in cervical cancer cells via endocytosis, resulting in a significant increase in accumulation of DOX in tumor cells. 253,276,277 Table 3

CONFLICT OF INTERESTS
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no new data were created or analyzed.